First-In-Human,Phase 1, Randomized,Dose-Escalation Trial with Recombinant Anti–IL-20 Monoclonal Antibody in Patients with Psoriasis

Background

The current trial was a first-in-human clinical trial evaluating the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary efficacy of the recombinant monoclonal anti−interleukin-20 (IL-20) antibody, NNC0109-0012, which targets the inflammatory cytokine IL-20.

Methods

In total, 48 patients aged 18 to 75 years with moderate to severe stable chronic plaque psoriasis with affected body surface area ≥15% and physician global assessment score ≥3 were enrolled in this randomized, double-blind, multicenter, placebo-controlled, phase 1 dose-escalation trial. Patients were randomized within each single dose cohort (0.01, 0.05, 0.2, 0.6, 1.5, or 3.0 mg/kg) or multiple dose cohort (0.05, 0.2, 0.5, 1.0, or 2.0 mg/kg; 1 dose every other week for 7 weeks) of NNC0109-0012 or placebo in a 3:1 ratio. In the expansion phase, 7 patients were randomized to weekly doses of 2.0 mg/kg NNC0109-0012 or placebo for 7 weeks. The primary objective, safety and tolerability, was assessed by evaluating adverse events (AEs). Additional endpoints included pharmacokinetics, pharmacodynamics, and clinical response (assessed using the Psoriasis Area and Severity Index [PASI] score).

Results

AEs were reported in 85% of patients (n = 40) in the initial study phases (NNC0109-0012, 83%; placebo, 92%) and in 4 of 7 patients in the multiple-dose expansion phase. One serious AE was reported but was judged not to be causally related to NNC0109-0012. No dose-limiting toxicities were reported. NNC0109-0012 pharmacokinetics was similar to other monoclonal antibodies, with an average half-life of approximately 3 weeks. There was a dose-proportional increase in area under the curve and maximum concentration after single dosing. No substantial changes in pharmacodynamic parameters were observed. The expansion phase was terminated early due to apparent lack of PASI improvement.

Conclusion

Single and multiple doses of NNC0109-0012, ranging from 0.05 to 3.0 mg/kg, were well tolerated in patients with psoriasis and exhibited pharmacokinetics similar to that of other monoclonal antibodies.

Trial Registration

ClinicalTrials.gov NCT01261767     

Disease in children infected with HIV in Abidjan,C?te d'Ivoire.

OBJECTIVE--To document the range of disease in African children infected with HIV. DESIGN--Necropsy results in consecutive children aged 1 month or more who were HIV positive and in children who were HIV negative for comparison; IgA western blots on serum samples from children under 2 years of age who were positive for HIV-1 to test the validity of routine HIV serology. SETTING--Largest hospital in Abidjan, Côte d''Ivoire. SUBJECTS--78 children who were HIV positive and 77 children who were HIV negative on whom a necropsy was performed; their median ages at death were 18 and 21 months respectively. 36 HIV positive children and 29 HIV negative children were 1-14 months old; 42 HIV positive and 48 HIV negative children were > or = 15 months old. MAIN OUTCOME MEASURES--Cause of death and prevalence of diseases confirmed pathologically. RESULTS--Respiratory tract infections were more common in HIV positive than in HIV negative children (73 (94%) v 52 (68%); P < 0.05), and were aetiologically heterogeneous. Pneumocystis carinii pneumonia was found in 11 out of 36 (31%) HIV positive children aged < 15 months, but in no HIV negative children. Among older children measles was more common in HIV positive children (8/42 (19%) v 2/48 (4%); P < 0.06). Pyogenic meningitis was present in similar proportions of HIV positive and HIV negative children aged < 15 months (7/36 (19%) and 7/29 (24%)). In HIV positive children tuberculosis (1/78), lymphocytic interstitial pneumonitis (1/78), and HIV encephalitis (2/78) were rare. CONCLUSIONS--There is greater overlap between diseases associated with HIV infection and other common health problems in African children than there is in adults. Compared with adults, HIV positive children had a high prevalence of P carinii pneumonia and a low prevalence of tuberculosis. Measles, but not malaria, was associated with HIV infection.     

Meteorological factors are associated with hemorrhagic fever with renal syndrome in Jiaonan County,China, 2006–2011

This study examined the effect of meteorological factors on the occurrence of hemorrhagic fever with renal syndrome (HFRS) using a generalized additive model with penalized smoothing splines in Jiaonan, China, from 2006 to 2011. The dose–response relationship was first examined, and then the association between daily meteorological variables and HFRS occurrence was investigated according to the dose–response curves. There were two linear segments in the temperature–HFRS relationship curve. When daily temperature was lower than 17 °C, a positive association was found [with excessive risk (ER) for 1 °C increase on the current day being 2.56 %, 95 % confidence interval (CI): 0.36 % to 4.80 %]. An inverse association was found when daily temperature was higher than 17 °C [ER for 1 °C increase on the current day was ?12.82 % (95 % CI: ?17.51 % to ?7.85 %)]. Inverse associations were observed for relative humidity [ER for 1 % increase on lag day 4 was ?1.21 % (95 % CI: ?1.63 % to ?0.79 %)] and rainfall [ER for 1 mm increase on lag day 1 was ?2.20 % (95 % CI: ?3.56 % to ?0.82 %)]. Meteorological factors might be important predictor of HFRS epidemics in Jiaonan County.     

Periodicity,persistence, and collapse in host–parasitoid systems with egg limitation

There is an emerging consensus that parasitoids are limited by the number of eggs which they can lay as well as the amount of time they can search for their hosts. Since egg limitation tends to destabilize host–parasitoid dynamics, successful control of insect pests by parasitoids requires additional stabilizing mechanisms such as heterogeneity in the distribution of parasitoid attacks and host density-dependence. To better understand how egg limitation, search limitation, heterogeneity in parasitoid attacks, and host density-dependence influence host–parasitoid dynamics, discrete time models accounting for these factors are analyzed. When parasitoids are purely egg-limited, a complete anaylsis of the host–parasitoid dynamics are possible. The analysis implies that the parasitoid can invade the host system only if the parasitoid’s intrinsic fitness exceeds the host’s intrinsic fitness. When the parasitoid can invade, there is a critical threshold, CV ^*>1, of the coefficient of variation (CV) of the distribution of parasitoid attacks that determines that outcome of the invasion. If parasitoid attacks sufficiently aggregated (i.e., CV>CV ^*), then the host and parasitoid coexist. Typically (in a topological sense), this coexistence is shown to occur about a periodic attractor or a stable equilibrium. If the parasitoid attacks are sufficiently random (i.e. CV<CV ^*), then the parasitoid drives the host to extinction. When parasitoids are weakly search-limited as well as egg-limited, coexistence about a global attractor occurs even if CV<CV ^*. However, numerical simulations suggest that the nature of this attractor depends critically on whether CV<1 or CV>1. When CV<1, the parasitoid exhibits highly oscillatory dynamics. Alternatively, when parasitoid attacks are sufficiently aggregated but not overly aggregated (i.e. CV>1 but close to 1), the host and parasitoid coexist about a stable equilibrium with low host densities. The implications of these results for classical biological control are discussed.     

Vegetation changes in semi-natural meadows with unchanged management in southern Sweden, 1965–1990

Vegetation records from 1963–66 and 1990 were compared in eight semi-permanent plots from continuously managed, semi-natural hay-meadows from five sites in southern Sweden to find out whether changes in plant communities could be detected in these grasslands over a period of ca. 25 years in spite of unchanged management. It was found that species richness, α-diversity and species frequency distribution remained fairly constant over the period, while there was considerable species turnover. However, it was also found that the total cover of vascular plants increased as well as the proportion of graminoids, both in cover and in species number, with a corresponding decrease of forbs. The number of species favoured by mowing also decreased as well as the number of typical grassland species. The detected changes are interpreted partly as effects of increased loads of atmospheric nitrogen deposition, and partly related to small changes in management intensity (changed frequency of mowing and/or aftermath grazing). It is possible that the significant land use changes in adjacent areas resulting in large landscape changes (increase of forests and woodlands) might have negatively influenced the interchange of grassland species, leading to an increase in the number of non-typical grassland species in the meadows.     

Out with the old,in with the new? Comparing methods for measuring protein degradation

Protein degradation is a critical factor in controlling cellular protein abundance. Here, we compare classical methods for determining protein degradation rates to a novel GFP (green fluorescent protein) fusion protein based method that assesses the intrinsic stability of cloned cDNA library products by flow cytometry [Yen et al. (2008) Science 322, 918]. While no method is perfect, we conclude that chimeric gene reporter approaches, though powerful, should be applied cautiously, due principally to GFP (or other reporter tag) interference with protein organelle targeting or incorporation into macromolecular assemblies, both of which cause spuriously high degradation rates.     

Brain-derived proteins in the CSF, do they correlate with brain pathology in CJD?

Background

Brain derived proteins such as 14-3-3, neuron-specific enolase (NSE), S 100b, tau, phosphorylated tau and Aβ_1–42 were found to be altered in the cerebrospinal fluid (CSF) in Creutzfeldt-Jakob disease (CJD) patients. The pathogenic mechanisms leading to these abnormalities are not known, but a relation to rapid neuronal damage is assumed. No systematic analysis on brain-derived proteins in the CSF and neuropathological lesion profiles has been performed.

Methods

CSF protein levels of brain-derived proteins and the degree of spongiform changes, neuronal loss and gliosis in various brain areas were analyzed in 57 CJD patients.

Results

We observed three different patterns of CSF alteration associated with the degree of cortical and subcortical changes. NSE levels increased with lesion severity of subcortical areas. Tau and 14-3-3 levels increased with minor pathological changes, a negative correlation was observed with severity of cortical lesions. Levels of the physiological form of the prion protein (PrP^c) and Aβ_1–42 levels correlated negatively with cortical pathology, most clearly with temporal and occipital lesions.

Conclusion

Our results indicate that the alteration of levels of brain-derived proteins in the CSF does not only reflect the degree of neuronal damage, but it is also modified by the localization on the brain pathology. Brain specific lesion patterns have to be considered when analyzing CSF neuronal proteins.     

Golimumab in patients with active rheumatoid arthritis after treatment with tumor necrosis factor α inhibitors: findings with up to five years of treatment in the multicenter,randomized, double-blind,placebo-controlled,phase 3 GO-AFTER study

IntroductionThe aim of this study was to assess long-term golimumab therapy in rheumatoid arthritis (RA) patients who discontinued previous tumor necrosis factor-α (TNF)-inhibitor(s).MethodsPatients enrolled into this multicenter, randomized, double-blind, placebo-controlled study of active RA (≥4 tender, ≥4 swollen joints) received placebo (Group 1) or golimumab 50 mg (Group 2) or 100 mg (Group 3) injections every 4 weeks. Patients in Groups 1 and 2 with inadequate response at week 16 escaped to golimumab 50 and 100 mg, respectively. At week 24, Group 1 patients crossed-over to golimumab 50 mg, Group 2 continued golimumab 50/100 mg per escape status, and Group 3 maintained dosing. During the long-term-extension (LTE), golimumab 50 mg could be increased to 100 mg, and 100 mg could be decreased to 50 mg. Data through 5 years are reported for all patients (safety) and patients using methotrexate (efficacy, intention-to-treat (ITT) analysis with last-observation-carried-forward for missing data and non-responder imputation for unsatisfactory efficacy discontinuations).ResultsIn total, 459 of 461 randomized patients received the study agent, 304 of whom were methotrexate-treated and included in efficacy analyses. Through week 256, the proportions of methotrexate-treated patients achieving American-College-of-Rheumatology (ACR) responses were 37.6% to 47.0% for ACR20, 21.4% to 35.0% for ACR50, and 7.8% to 17.0% for ACR70 response across randomized groups. Golimumab safety through week 268 was generally consistent with that at week 24 and week 160 and other anti-TNF agents.ConclusionsIn some patients with active RA discontinuing previous TNF-antagonist therapy, golimumab safety and efficacy, assessed conservatively with ITT analyses, was confirmed through 5 years.

Trial registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00299546","term_id":"NCT00299546"}}NCT00299546. Registered 03 March 2006.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0516-6) contains supplementary material, which is available to authorized users.     

Tiotropium Respimat? in asthma: a double-blind,randomised, dose-ranging study in adult patients with moderate asthma

Background

Tiotropium, a once-daily long-acting anticholinergic bronchodilator, when administered via Respimat® SoftMist™ inhaler (tiotropium Respimat®) significantly reduces the risk of severe exacerbations and improves lung function in patients with severe persistent asthma that is not fully controlled despite using inhaled corticosteroids (ICS) and long-acting β₂-agonists. To further explore the dose–response curve in asthma, we investigated the efficacy and safety of three different doses of tiotropium Respimat® as add-on to ICS in symptomatic patients with moderate persistent asthma.

Methods

In this randomised, double-blind, placebo-controlled, four-way crossover study, patients were randomised to tiotropium Respimat® 5 μg, 2.5 μg or 1.25 μg or placebo Respimat®, once daily in the evening. Each treatment was administered for 4 weeks, without washout between treatment periods. Eligibility criteria included ≥60% and ≤90% of predicted normal forced expiratory volume in 1 second (FEV₁) and seven-question Asthma Control Questionnaire mean score of ≥1.5. Patients were required to continue maintenance treatment with stable medium-dose ICS for at least 4 weeks prior to and during the treatment period. Long-acting β₂-agonists were not permitted during the treatment phase. The primary efficacy end point was peak FEV₁ measured within 3 hours after dosing (peak FEV_1(0-3h)) at the end of each 4-week period, analysed as a response (change from study baseline).

Results

In total, 149 patients were randomised and 141 completed the study. Statistically significant improvements in peak FEV_1(0-3h) response were observed with each tiotropium Respimat® dose versus placebo (all P < 0.0001). The largest difference from placebo was with tiotropium Respimat® 5 μg (188 mL). Trough FEV₁ and FEV₁ area under the curve (AUC)_(0-3h) responses were greater with each tiotropium Respimat® dose than with placebo (all P < 0.0001), and both were greatest with 5 μg. Peak forced vital capacity (FVC)_(0-3h), trough FVC and FVC AUC_(0-3h) responses, versus placebo, were greatest with tiotropium Respimat® 5 μg (P < 0.0001, P = 0.0012 and P < 0.0001, respectively). Incidence of adverse events was comparable between placebo and all tiotropium Respimat® groups.

Conclusions

Once-daily tiotropium Respimat® add-on to medium-dose ICS improves lung function in symptomatic patients with moderate asthma. Overall, improvements were largest with tiotropium Respimat® 5 μg.

Trial registration

ClinicalTrials.gov identifier {"type":"clinical-trial","attrs":{"text":"NCT01233284","term_id":"NCT01233284"}}NCT01233284.     

Genetic variation in piñon pine, Pinus edulis, associated with summer precipitation

Three previous reports of microgeographical variation of glycerate dehydrogenase (Gly) frequencies in piñon, Pinus edulis, established the hypothesis that Gly frequencies contribute to adaptation to heterogeneous environments, specifically to variation in soil moisture. In each of these studies, the frequency of the Gly‐3 allele or of Gly‐33 homozygotes was higher on dry sites than on nearby moist sites. Here we attempt to extend these observations by testing the hypothesis that Gly frequencies respond to soil moisture variation on a range‐wide scale. Gly frequencies were surveyed in 11 natural populations, and the frequency of the Gly‐3 allele varied from 0.27 to 0.65 among the sample sites. Elevation varied from 1650 to 3100 m, and summer precipitation, defined as precipitation from April to August, varied from 13.7 to 26.4 cm. The soil types at the collection sites were schist, quaternary volcanic or a mixture of shale and sandstone. Logistic regression revealed that Gly frequencies did not respond to either elevation or soil type, but were related to summer precipitation (P < 0.01). The correlation between summer precipitation and the frequency of the Gly‐3 allele was r = ?0.92 (P < 0.001). Thus, the patterns of differentiation on microgeographical scales are consistent with greater differentiation on a range‐wide scale.     

NMR assignment of polymerase β labeled with 2H, 13C, and 15N in complex with substrate DNA

DNA Polymerase β is a multifunctional enzyme involved in base excision repair of nuclear DNA in vertebrate cells. We present here the first assignments of the full-length protein (335 residues, 39 kDa) in the presence of a double gap—double hairpin DNA (22 nucleotides, 7 kDa).     

Fluorescence quenching with lindane in small unilamellar l,α-dimyristoylphosphatidylcholine vesicles

The lateral mobility and lipid-water partition of the pesticide lindane was studied by fluorescence quenching of N-isopropylcarbazole (NIPC) and l,-palmitoyl--(N-carbazolyl) undecanoylphosphatidylcholine (PCUPC) in liposomes of dimyristoylphosphatidylcholine at 50°C. In isotropic solvents the quenching reaction was highly inefficient. A scheme for dynamic quenching, in which the monomolecular quenching rate constant is small, was valid. In lipid bilayers the same scheme was applied to describe the quenching results but the rate constant of the backreaction of the excited complex to quencher and excited probe was of comparable magnitude to the monomolecular quenching rate constant. This phenomenon results in biexponential decays of the fluorescent probe in the presence of quencher. All the rate constants of the scheme could be determined. Stern-Volmer plots at different membrane concentrations were obtained from fluorescence intensity and decay time measurements. From these plots the true bimolecular quenching rate constant, k _q , and the rate constant for lateral diffusion, k _d , were determined: . The smaller value of k _q compared to k _d for the quenching reaction of NIPC with lindane indicates that this quenching reaction is not diffusion controlled. The lateral diffusion coefficient D of lindane was found to be 1.7±0.2×10^-6 cm²/s in dimyristoylphosphatidylcholine vesicles at 50°C. The partition coefficient of lindane in these lipid bilayers is very high (>2000).Abbreviations DMPC dimyristoylphosphatidylcholine - lindane 1,2,3,4,5,6-hexachlorocyclohexane (-isomer) - NIPC N-isopropylcarbazole - PCUPC l,-palmitoyl--(N-carbazolyl) undecanoylphosphatidylcholine - SUV small unilamellar vesicles     

Changes in HIF-1α protein, pyruvate dehydrogenase phosphorylation, and activity with exercise in acute and chronic hypoxia

Exercise under acute hypoxia elicits a large increase in blood lactate concentration ([La](b)) compared with normoxic exercise. However, several studies in humans show that with the transition to chronic hypoxia, exercise [La](b) returns to normoxic levels. Although extensively examined over the last decades, the muscle-specific mechanisms responsible for this phenomenon remain unknown. To assess the changes in skeletal muscle associated with a transition from acute to chronic hypoxia, CD-1 mice were exposed for 24 h (24H), 1 wk (1WH), or 4 wk (4WH) to hypobaric hypoxia (equivalent to 4,300 m), exercised under 12% O(2), and compared with normoxic mice (N) at 21% O(2). Since the enzyme pyruvate dehydrogenase (PDH) plays a major role in the metabolic fate of pyruvate (oxidation vs. lactate production), we assessed the changes in its activity and regulation. Here we report that when run under hypoxia, 24H mice exhibited the highest blood and intramuscular lactate of all groups, while the 1WH group approached N group values. Concomitantly, the 24H group exhibited the lowest PDH activity, associated with a higher phosphorylation (inactive) state of the Ser(232) residue of PDH, a site specific to PDH kinase-1 (PDK1). Furthermore, protein levels of PDK1 and its regulator, the hypoxia inducible factor-1α (HIF-1α), were both elevated in the 24H group compared with N and 1WH groups. Overall, our results point to a novel mechanism in muscle where the HIF-1α pathway is desensitized in the transition from acute to chronic hypoxia, leading to a reestablishment of PDH activity and a reduction in lactate production by the exercising muscles.     

Cryptosporidiosis in children from 1 to 2 years of age, with acute diarrhea in Belém, Pará, Brazil

Two hundred and one samples obtained from 61 children were examined for Cryptosporidium infection during a period of 12 months. One hundred fifteen specimens were collected during diarrhoea episodes and the remaining 86 obtained out of diarrhoea period, as controls. All samples were examined by a modified Ziehl-Neelsen staining method. Cryptosporidium was detected in 6 (5.2%) of 115 samples from diarrhoeic children. All non-diarrhoeic control patients were negative for Cryptosporidium. The present study suggests that Cryptosporidium is an agent of self-limited diarrhoea among immunocompetent children from Belém, Pará.     

Treatment with PBI-4050 in patients with Alström syndrome: study protocol for a phase 2, single-Centre,single-arm,open-label trial

Background

Alström syndrome (ALMS) is a very rare autosomal recessive monogenic disorder caused by a mutation in the ALMS1 gene and characterised by childhood onset obesity, dyslipidaemia, advanced non-alcoholic fatty liver disease, diabetes and extreme insulin resistance. There is evidence of multi-organ fibrosis in ALMS and severity of the disease often leads to organ failure with associated morbidities, resulting in reduced life expectancy. There are no specific treatments for this disease, and current management consists of only symptomatic therapies. PBI-4050 is a new molecular entity with demonstrated anti-inflammatory and anti-fibrotic activities in preclinical models, including animal models of human diseases characterized by progressive fibrosis in the kidney, heart, liver and lungs. Moreover, completed Phase 2 studies in type 2 diabetes mellitus with metabolic syndrome and idiopathic pulmonary fibrosis further support the anti-inflammatory and anti-fibrotic activity of PBI-4050. Together, these data suggest that PBI-4050 has the potential to treat the pathological inflammatory and fibrotic features of ALMS. The aim of this study is to evaluate the safety and anti-inflammatory & anti-fibrotic activities of PBI-4050 in subjects with ALMS.

Methods

This is a Phase 2, single-centre, single-arm, open-label trial. A total of 18 patients with ALMS will be enrolled to receive PBI-4050 at a total daily oral dose of 800?mg for an initial 24?weeks with continuation for an additional 36 or 48?weeks. Standard assessments of safety include adverse events, clinical laboratory tests, vital signs, physical examination and electrocardiograms. Efficacy assessments include adipose tissue biopsy, hyperinsulinaemic-euglycaemic glucose clamp, adipose tissue microdialysis, liver transient elastography, liver and cardiac magnetic resonance imaging, and laboratory blood tests.

Discussion

This is the first clinical study of PBI-4050 in subjects with ALMS. Given the rarity and complexity of the disease, a single-centre, single-arm, open-label design has been chosen to maximise subject exposure and increase the likelihood of achieving our study endpoints. The results will provide valuable safety and preliminary evidence of the effects of PBI-4050 in ALMS, a rare heterogeneous disease associated with progressive fibrosis and premature mortality.

Trial registration

The trial is registered on ClinicalTrials.gov (Identifier; NCT02739217, February 2016) and European Union Drug Regulating Authorities Clinical Trials (EudraCT Number 2015–001625-16, Sept 2015).

     

G-11 staining in Turner's syndrome with mos 45,X/46,X,r(?)

Mos 45,X/46,X,r(?) in 4 patients with Turner's syndrome and no signs of virilization, and in one pair of monozygotic twins, one of them with clitoral hypertrophy, was studied using combined cytogenetic techniques and specially G-11 staining for the characterization of the X or Y origin of the rings. In all 6 patients the ring was G-11 positive, attesting its Y origin. Both twins were operated and bilateral streak gonads with a bilateral nodule of testicular tissue were found. Similar small rings were also studied in one patient with mos 46,XX/46,X,r(X) and in one nonvirilized Turner's syndrome patient with a larger ring; in these two cases the ring was G-11 negative. It seems that the small rings occasionally found in Turner's syndrome are more frequently from Y origin and therefore prophylactic gonadectomy should be considered.     

Polyparasitism with Schistosoma mansoni,geohelminths, and intestinal protozoa in rural Côte d'Ivoire

Single species infections with schistosomes, geohelminths, and intestinal protozoans are common over large parts of sub-Saharan Africa, and it is expected that polyparasitism affects a considerable proportion of the population, hence posing a great toll on public health. However, few investigations have been carried out to quantify the extent of polyparasitism. Here, a detailed assessment is reported for the epidemiology of Schistosoma mansoni, geohelminths, and intestinal protozoan infections, with particular emphasis on polyparasitism among 260 community members in rural C?te d'Ivoire. Schistosoma mansoni, Entamoeba coli, and hookworm were the predominant species with prevalences of 71.5, 64.6, and 51.9%, respectively. Only 8 individuals displayed no infection, whereas two-thirds of the population harbored 3 or more parasites concurrently. There were a series of significant pairwise parasite co-occurrences, e.g., between S. mansoni and hookworms and between S. mansoni and E. coli. It is concluded that polyparasitism in the population studied here was very common, which is probably the case also in other areas of rural C?te d'Ivoire and elsewhere in sub-Saharan Africa. These findings call for integrated approaches to effectively control multiple parasitic and protozoan infections.